Fringe is a glycosyltransferase that modifies Notch

Notch receptors function in highly conserved intercellular signalling pathways that direct cell-fate decisions, proliferation and apoptosis in metazoans. Fringe proteins can positively and negatively modulate the ability of Notch ligands to activate the Notch receptor. Here we establish the biochemical mechanism of Fringe action. Drosophila and mammalian Fringe proteins possess a fucose-specific beta1,3 N-acetylglucosaminyltransferase activity that initiates elongation of O-linked fucose residues attached to epidermal growth factor-like sequence repeats of Notch. We obtained biological evidence that Fringe-dependent elongation of O-linked fucose on Notch modulates Notch signalling by using co-culture assays in mammalian cells and by expression of an enzymatically inactive Fringe mutant in Drosophila. The post-translational modification of Notch by Fringe represents a striking example of modulation of a signalling event by differential receptor glycosylation and identifies a mechanism that is likely to be relevant to other signalling pathways.     

A tension-induced mechanotransduction pathway promotes epithelial morphogenesis

Mechanotransduction refers to the transformation of physical forces into chemical signals. It generally involves stretch-sensitive channels or conformational change of cytoskeleton-associated proteins. Mechanotransduction is crucial for the physiology of several organs and for cell migration. The extent to which mechanical inputs contribute to development, and how they do this, remains poorly defined. Here we show that a mechanotransduction pathway operates between the body-wall muscles of Caenorhabditis elegans and the epidermis. This pathway involves, in addition to a Rac GTPase, three signalling proteins found at the hemidesmosome: p21-activated kinase (PAK-1), the adaptor GIT-1 and its partner PIX-1. The phosphorylation of intermediate filaments is one output of this pathway. Tension exerted by adjacent muscles or externally exerted mechanical pressure maintains GIT-1 at hemidesmosomes and stimulates PAK-1 activity through PIX-1 and Rac. This pathway promotes the maturation of a hemidesmosome into a junction that can resist mechanical stress and contributes to coordinating the morphogenesis of epidermal and muscle tissues. Our findings suggest that the C. elegans hemidesmosome is not only an attachment structure, but also a mechanosensor that responds to tension by triggering signalling processes. We suggest that similar pathways could promote epithelial morphogenesis or wound healing in other organisms in which epithelial cells adhere to tension-generating contractile cells.     

Structural basis for EGFR ligand sequestration by Argos

Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes. Inappropriate activation of these receptors is a key feature of many human cancers, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signalling, called Argos, was identified in Drosophila. We showed previously that Argos functions by directly binding (and sequestering) growth factor ligands that activate EGFR. Here we describe the 1.6-A resolution crystal structure of Argos bound to an EGFR ligand. Contrary to expectations, Argos contains no EGF-like domain. Instead, a trio of closely related domains (resembling a three-finger toxin fold) form a clamp-like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF. The individual Argos domains share unexpected structural similarities with the extracellular ligand-binding regions of transforming growth factor-beta family receptors. The three-domain clamp of Argos also resembles the urokinase-type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF-like module of uPA. Our results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues. In addition, the structures presented here define requirements for the design of artificial EGF-sequestering proteins that would be valuable anti-cancer therapeutics.     

Argos inhibits epidermal growth factor receptor signalling by ligand sequestration

The epidermal growth factor receptor (EGFR) has critical functions in development and in many human cancers. During development, the spatial extent of EGFR signalling is regulated by feedback loops comprising both well-understood activators and less well-characterized inhibitors. In Drosophila melanogaster the secreted protein Argos functions as the only known extracellular inhibitor of EGFR, with clearly identified roles in multiple stages of development. Argos is only expressed when the Drosophila EGFR (DER) is activated at high levels, and downregulates further DER signalling. Although there is ample genetic evidence that Argos inhibits DER activation, the biochemical mechanism has not been established. Here we show that Argos inhibits DER signalling without interacting directly with the receptor, but instead by sequestering the DER-activating ligand Spitz. Argos binds tightly to the EGF motif of Spitz and forms a 1:1 (Spitz:Argos) complex that does not bind DER in vitro or at the cell surface. Our results provide an insight into the mechanism of Argos function, and suggest new strategies for EGFR inhibitor design.     

Cytotoxic T-lymphocyte activation involves a cascade of signalling and adhesion events.

In addition to the antigen-specific T-cell receptor (TCR), T cells bear an array of 'accessory' molecules that can contribute to stable adhesion to the antigen-bearing cell and provide costimulatory signals. For several of these, T-cell adhesion to the ligand can be activated by TCR-dependent signalling (a signal from the TCR primes the coreceptor to bind to its ligand). It is unclear whether the individual coreceptors share common mechanisms of priming and cosignalling, and perhaps act in a redundant manner, or whether they act in a distinct way and contribute uniquely to the activation process. We report here the use of isolated alloantigen, class I proteins and fibronectin ligands to show that coreceptors on cytotoxic T lymphocytes are activated sequentially and deliver distinct biochemical signals on binding to their ligands. TCR engagement activates CD8 by a protein tyrosine kinase-dependent pathway, and CD8 then acts as a signal for initiation of polyphosphoinositide hydrolysis on binding to class I. In contrast, activated adhesion to fibronectin does not initiate polyphosphoinositide hydrolysis, but amplifies hydrolysis once it has been initiated. Thus, cytotoxic T-lymphocyte activation involves a TCR-initiated cascade of adhesion and signalling events leading to response.     

EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF

Cross-communication between different signalling systems allows the integration of the great diversity of stimuli that a cell receives under varying physiological situations. The transactivation of epidermal growth factor receptor (EGFR)-dependent signalling pathways upon stimulation of G-protein-coupled receptors (GPCRs), which are critical for the mitogenic activity of ligands such as lysophosphatidic acid, endothelin, thrombin, bombesin and carbachol, provides evidence for such an interconnected communication network. Here we show that EGFR transactivation upon GPCR stimulation involves proHB-EGF and a metalloproteinase activity that is rapidly induced upon GPCR-ligand interaction. We show that inhibition of proHB-EGF processing blocks GPCR-induced EGFR transactivation and downstream signals. The pathophysiological significance of this mechanism is demonstrated by inhibition of constitutive EGFR activity upon treatment of PC3 prostate carcinoma cells with the metalloproteinase inhibitor batimastat. Together, our results establish a new mechanistic concept for cross-communication among different signalling systems.     

Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors

Mutationally activated kinases define a clinically validated class of targets for cancer drug therapy. However, the efficacy of kinase inhibitors in patients whose tumours harbour such alleles is invariably limited by innate or acquired drug resistance. The identification of resistance mechanisms has revealed a recurrent theme—the engagement of survival signals redundant to those transduced by the targeted kinase. Cancer cells typically express multiple receptor tyrosine kinases (RTKs) that mediate signals that converge on common critical downstream cell-survival effectors—most notably, phosphatidylinositol-3-OH kinase (PI(3)K) and mitogen-activated protein kinase (MAPK). Consequently, an increase in RTK-ligand levels, through autocrine tumour-cell production, paracrine contribution from tumour stroma or systemic production, could confer resistance to inhibitors of an oncogenic kinase with a similar signalling output. Here, using a panel of kinase-'addicted' human cancer cell lines, we found that most cells can be rescued from drug sensitivity by simply exposing them to one or more RTK ligands. Among the findings with clinical implications was the observation that hepatocyte growth factor (HGF) confers resistance to the BRAF inhibitor PLX4032 (vemurafenib) in BRAF-mutant melanoma cells. These observations highlight the extensive redundancy of RTK-transduced signalling in cancer cells and the potentially broad role of widely expressed RTK ligands in innate and acquired resistance to drugs targeting oncogenic kinases.     

Delta-promoted filopodia mediate long-range lateral inhibition in Drosophila

Drosophila thoracic mechanosensory bristles originate from cells that are singled out from 'proneural' groups of competent epithelial cells. Neural competence is restricted to individual sensory organ precursors (SOPs) by Delta/Notch-mediated 'lateral inhibition', whereas other cells in the proneural field adopt an epidermal fate. The precursors of the large macrochaetes differentiate separately from individual proneural clusters that comprise about 20-30 cells or as heterochronic pairs from groups of more than 100 cells, whereas the precursors of the small regularly spaced microchaetes emerge from even larger proneural fields. This indicates that lateral inhibition might act over several cell diameters; it was difficult to reconcile with the fact that the inhibitory ligand Delta is membrane-bound until the observation that SOPs frequently extend thin processes offered an attractive hypothesis. Here we show that the extension of these planar filopodia--a common attribute of wing imaginal disc cells--is promoted by Delta and that their experimental suppression reduces Notch signalling in distant cells and increases bristle density in large proneural groups, showing that these membrane specializations mediate long-range lateral inhibition.     

VEGF regulates haematopoietic stem cell survival by an internal autocrine loop mechanism

Vascular endothelial growth factor (VEGF) is a principal regulator of blood vessel formation and haematopoiesis, but the mechanisms by which VEGF differentially regulates these processes have been elusive. Here we describe a regulatory loop by which VEGF controls survival of haematopoietic stem cells (HSCs). We observed a reduction in survival, colony formation and in vivo repopulation rates of HSCs after ablation of the VEGF gene in mice. Intracellularly acting small-molecule inhibitors of VEGF receptor (VEGFR) tyrosine kinase dramatically reduced colony formation of HSCs, thus mimicking deletion of the VEGF gene. However, blocking VEGF by administering a soluble VEGFR-1, which acts extracellularly, induced only minor effects. These findings support the involvement in HSC survival of a VEGF-dependent internal autocrine loop mechanism (that is, the mechanism is resistant to inhibitors that fail to penetrate the intracellular compartment). Not only ligands selective for VEGF and VEGFR-2 but also VEGFR-1 agonists rescued survival and repopulation of VEGF-deficient HSCs, revealing a function for VEGFR-1 signalling during haematopoiesis.     

BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module

Engulfment and subsequent degradation of apoptotic cells is an essential step that occurs throughout life in all multicellular organisms. ELMO/Dock180/Rac proteins are a conserved signalling module for promoting the internalization of apoptotic cell corpses; ELMO and Dock180 function together as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac, and thereby regulate the phagocyte actin cytoskeleton during engulfment. However, the receptor(s) upstream of the ELMO/Dock180/Rac module are still unknown. Here we identify brain-specific angiogenesis inhibitor 1 (BAI1) as a receptor upstream of ELMO and as a receptor that can bind phosphatidylserine on apoptotic cells. BAI1 is a seven-transmembrane protein belonging to the adhesion-type G-protein-coupled receptor family, with an extended extracellular region and no known ligands. We show that BAI1 functions as an engulfment receptor in both the recognition and subsequent internalization of apoptotic cells. Through multiple lines of investigation, we identify phosphatidylserine, a key 'eat-me' signal exposed on apoptotic cells, as a ligand for BAI1. The thrombospondin type 1 repeats within the extracellular region of BAI1 mediate direct binding to phosphatidylserine. As with intracellular signalling, BAI1 forms a trimeric complex with ELMO and Dock180, and functional studies suggest that BAI1 cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. Last, decreased BAI1 expression or interference with BAI1 function inhibits the engulfment of apoptotic targets ex vivo and in vivo. Thus, BAI1 is a phosphatidylserine recognition receptor that can directly recruit a Rac-GEF complex to mediate the uptake of apoptotic cells.     

Crystal structure of an Eph receptor-ephrin complex.

The Eph family of receptor tyrosine kinases and their membrane-anchored ephrin ligands are important in regulating cell-cell interactions as they initiate a unique bidirectional signal transduction cascade whereby information is communicated into both the Eph-expressing and the ephrin-expressing cells. Initially identified as regulators of axon pathfinding and neuronal cell migration, Ephs and ephrins are now known to have roles in many other cell-cell interactions, including those of vascular endothelial cells and specialized epithelia. Here we report the crystal structure of the complex formed between EphB2 and ephrin-B2, determined at 2.7 A resolution. Each Eph receptor binds an ephrin ligand through an expansive dimerization interface dominated by the insertion of an extended ephrin loop into a channel at the surface of the receptor. Two Eph-Ephrin dimers then join to form a tetramer, in which each ligand interacts with two receptors and each receptor interacts with two ligands. The Eph and ephrin molecules are precisely positioned and orientated in these complexes, promoting higher-order clustering and the initiation of bidirectional signalling.     

Two distinct modes of guidance signalling during collective migration of border cells

Although directed migration is a feature of both individual cells and cell groups, guided migration has been studied most extensively for single cells in simple environments. Collective guidance of cell groups remains poorly understood, despite its relevance for development and metastasis. Neural crest cells and neuronal precursors migrate as loosely organized streams of individual cells, whereas cells of the fish lateral line, Drosophila tracheal tubes and border-cell clusters migrate as more coherent groups. Here we use Drosophila border cells to examine how collective guidance is performed. We report that border cells migrate in two phases using distinct mechanisms. Genetic analysis combined with live imaging shows that polarized cell behaviour is critical for the initial phase of migration, whereas dynamic collective behaviour dominates later. PDGF- and VEGF-related receptor and epidermal growth factor receptor act in both phases, but use different effector pathways in each. The myoblast city (Mbc, also known as DOCK180) and engulfment and cell motility (ELMO, also known as Ced-12) pathway is required for the early phase, in which guidance depends on subcellular localization of signalling within a leading cell. During the later phase, mitogen-activated protein kinase and phospholipase Cgamma are used redundantly, and we find that the cluster makes use of the difference in signal levels between cells to guide migration. Thus, information processing at the multicellular level is used to guide collective behaviour of a cell group.     

Endocytosis-mediated downregulation of LIN-12/Notch upon Ras activation in Caenorhabditis elegans

The coordination of signals from different pathways is important for cell fate specification during animal development. Here, we define a novel mode of crosstalk between the epidermal growth factor receptor/Ras/mitogen-activated protein kinase cascade and the LIN-12/Notch pathway during Caenorhabditis elegans vulval development. Six vulval precursor cells (VPCs) are initially equivalent but adopt different fates as a result of an inductive signal mediated by the Ras pathway and a lateral signal mediated by the LIN-12/Notch pathway. One consequence of activating Ras is a reduction of LIN-12 protein in P6.p (ref. 2), the VPC believed to be the source of the lateral signal. Here we identify a 'downregulation targeting signal' (DTS) in the LIN-12 intracellular domain, which encompasses a di-leucine-containing endocytic sorting motif. The DTS seems to be required for internalization of LIN-12, and on Ras activation it might mediate altered endocytic routing of LIN-12, leading to downregulation. We also show that if LIN-12 is stabilized in P6.p, lateral signalling is compromised, indicating that LIN-12 downregulation is important in the appropriate specification of cell fates in vivo.     

Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signalling

Developing tissues and growing tumours produce vascular endothelial growth factors (VEGFs), leading to the activation of the corresponding receptors in endothelial cells. The resultant angiogenic expansion of the local vasculature can promote physiological and pathological growth processes. Previous work has uncovered that the VEGF and Notch pathways are tightly linked. Signalling triggered by VEGF-A (also known as VEGF) has been shown to induce expression of the Notch ligand DLL4 in angiogenic vessels and, most prominently, in the tip of endothelial sprouts. DLL4 activates Notch in adjacent cells, which suppresses the expression of VEGF receptors and thereby restrains endothelial sprouting and proliferation. Here we show, by using inducible loss-of-function genetics in combination with inhibitors in vivo, that DLL4 protein expression in retinal tip cells is only weakly modulated by VEGFR2 signalling. Surprisingly, Notch inhibition also had no significant impact on VEGFR2 expression and induced deregulated endothelial sprouting and proliferation even in the absence of VEGFR2, which is the most important VEGF-A receptor and is considered to be indispensable for these processes. By contrast, VEGFR3, the main receptor for VEGF-C, was strongly modulated by Notch. VEGFR3 kinase-activity inhibitors but not ligand-blocking antibodies suppressed the sprouting of endothelial cells that had low Notch signalling activity. Our results establish that VEGFR2 and VEGFR3 are regulated in a highly differential manner by Notch. We propose that successful anti-angiogenic targeting of these receptors and their ligands will strongly depend on the status of endothelial Notch signalling.     

Molecular and biological characterization of a murine ligand for CD40.

The CD40 surface molecule is a 277-amino-acid glycoprotein expressed on B lymphocytes, epithelial cells and some carcinoma cell lines. Monoclonal antibodies against CD40 mediate a variety of effects on B lymphocytes, including induction of intercellular adhesion, short- and long-term proliferation, differentiation and enhanced tyrosine phosphorylation of proteins. In addition, germinal centre centrocytes are prevented from undergoing apoptosis by activation through CD40 and receptor for antigen. These data indicate that CD40 could be a receptor for an unknown ligand with important functions in B-cell development and activation. This hypothesis is strengthened by the homology of the extracellular region of the CD40 molecule with a family of cell-surface glycoproteins that includes the receptors for nerve growth factor and tumour necrosis factor. Here we report the cloning of a ligand for CD40 that is expressed on the cell surface of activated T cells and mediates B-cell proliferation in the absence of co-stimulus, as well as IgE production in the presence of interleukin-4.     

Functional cloning of ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1

The leukocyte adhesion molecule LFA-1 mediates a wide range of lymphocyte, monocyte, natural killer cell, and granulocyte interactions with other cells in immunity and inflammation. LFA-1 (CD11a/CD18) is a receptor for intercellular adhesion molecule 1 (ICAM-1, CD54), a surface molecule which is constitutively expressed on some tissues and induced on other in inflammation. Induction of ICAM-1 on epithelial cells, endothelial cells and fibroblasts mediates LFA-1-dependent adhesion of lymphocytes. Several lines of evidence have suggested the existence of a second LFA-1 ligand: homotypic adhesion of one cell line was inhibited by a monoclonal antibody to LFA-1, but not by one to ICAM-1; there exists an LFA-1-dependent, ICAM-1-independent pathway of adhesion to endothelial cells; and also, there are some types of target cells in which LFA-1-dependent T-lymphocyte adhesion and lysis are independent of ICAM-1. We have cloned this second ligand, designated ICAM-2, using a novel method for identifying ligands of adhesion molecules. ICAM-2 is an integral membrane protein with two immunoglobulin-like domains, whereas ICAM-1 has five. Remarkably, ICAM-2 is much more closely related to the two most N-terminal domains of ICAM-1 (34% identity) than either ICAM-1 or ICAM-2 is to other members of the immunoglobulin superfamily, demonstrating the existence of a subfamily of immunoglobulin-like ligands that bind the same integrin receptor.     

免疫激活:关于toll样受体与nod样受体的探索

toll样受体和nod受体在天然免疫应答中是重要的家族。激发配体促进受体相互作用的精确检测从而导致多重信号的形成和多级信号转导的触发。这个过程能导致促炎因子的上调、细胞凋亡和调控其他免疫防御。最近,在这些受体家族中识别激活配体和配体的分子基础的检测上有重大进展。理解这些过程,对于新的疫苗佐剂的开发、感染性疾病的治疗、炎症反应和潜在的疾病甚至癌症,都提供了必要的信息。